Treatment of alphavirus-induced inflammation

ABSTRACT

Disclosed is a method of treating alphavirus infections, particularly in humans, in which pentosan polysulfate is administered to an infected subject. Whilst not effecting the viral load in a subject, the pentosan polysulfate acts to reduce inflammation in tissues, such as the muscles, and in the joints of a subject. In addition, cartilage damage in the joints may be reduced. The reduction in inflammation and/or cartilage damage acts to reduce the severe pain experienced by subjects suffering from alphavirus infections, such as Ross River virus, chikungunya virus and Barmah Forest virus.

The present application is a continuation of U.S. Pat. Application No.16/884,628. filed on May 27, 2020, which was a continuation of U.S. Pat.Application No. 16/304,550, filed on Nov. 26, 2018, which was a U.S.National Stage Application under 35 U.S.C. 371 of PCT/AU2016/050408,filed on May 26, 2016, each of which is incorporated herein byreference.

TECHNICAL FIELD

This invention relates to the treatment of animals, especially humans,infected with alphaviruses and more particularly to a treatment directedto the reduction of inflammation and/or cartilage damage arising fromthe infection.

SEQUENCE LISTING

This application contains a sequence listing which is incorporatedherein by reference in ST.26 XML format named 00265-026.PCT.US.CON2.xml,created Aug. 2, 2023, and is 11.3KB in size. The sequences contained inthe sequence listing are found throughout the originally filedapplication.

BACKGROUND

Arthropod-bome arthritogenic alphaviruses such as Ross River virus (RRV)and chikungunya virus (CHIKV) cause large epidemics of severemusculoskeletal disease. They have been progressively expanding theirglobal distribution, regularly emerging in new regions of the world (1,2). The hallmark of alphavirus disease is crippling joint pain andarthritis, which often has an extended duration leaving patientsbed-ridden and incapacitated. In 2014-2015, CHIKV further expanded itsglobal distribution by entering the Americas and is circulating inseveral Caribbean islands. As of 23^(rd) May 2016, the Pan AmericanHealth Organization (PAHO) reported an estimated total of over 1.5million cases since 2014, with 100,000 reported in 2016 so far,additionally, the first report of local autochthonous CHIKV transmissionin mainland USA was reported in July 2014 (3, 4). Due to the expandingrange of alphaviral infections, understanding the mechanisms by whichalphaviruses cause debilitating arthritic disease has becomeincreasingly important, especially as there are no specific treatmentsavailable (5).

The severe arthralgia/arthritis in the joints caused by alphaviruses canbe both acute and chronic. Ultrasonography of CHIKV patients with jointpain reveals striking tenosynovitis, bone erosion and synovialthickening (6). RRV antigen has been detected by immunofluorescence insynovial monocytes and macrophages during the early phase of illness(7), and in basal epidermal and eccrine duct epithelia three days afterthe onset of RRV exanthem (8). Using antigen staining and RT-PCR. RRVhas also been detected in synovial effusions more than one month afterthe onset of symptoms, providing evidence of persistent infection in theinflamed synovium (9).

The synovial space of joints is glycan-rich, containing high levels ofglycosaminoglycan (GAGs) frequently linked to protein backbones thatform proteoglycan structures. Chondrocytes are the major cell typeproducing the matrix of articular cartilage that is rich inproteoglycans (14). However, there have been no studies to elucidate theimpact of alphaviruses on cartilage and the proteoglycan matrix of thejoint.

In the work described herein, we show RRV infection results in similarhistopathology of the joint to that observed in rheumatoid arthritis(RA). This includes pannus-like formation, immune infiltration andcartilage damage. We further show that treatment with pentosanpolysulfate (PPS) ameliorates the severity of both RRV and CHIKVclinical disease, overall reduction in both immune infiltrates andsoluble pro- inflammatory factors. We also observed a change in thekinetics of the soluble factors involved in macrophage activation. InRRV-infection treatment also reduced the loss of articular cartilage andprotected the level of proteoglycans in the cartilage matrix, alteringthe expression of cartilage components including aggrecan (SEQ. ID. NO.2) and collagen in. Overall we show that PPS is a safe and effectivetreatment for both acute and chronic RRV-infection.

SUMMARY

Accordingly the present invention consists in a method of treating asubject having an alphavirus infection comprising administeringparenterally an amount of pentosan polysulfate or a salt thereofeffective to reduce alphavirus induced inflammation and/or alphavirusinduced cartilage damage in the subject.

In another aspect, the present invention consists in the use of pentosanpolysulfate or a salt thereof in the preparation of a medicament for thetreatment of alphavirus induced inflammation and/or to reduce alphavirusinduced cartilage damage in a subject having an alphavirus infection.

In a further aspect, the present invention consists in a compositioncomprising pentosan polysulfate and a pharmaceutically acceptablecarrier for use in treating alphavirus induced inflammation and/or toreducing alphavirus induced cartilage damage in a subject having analphavirus infection.

Whilst the subject to be treated may be an animal, preferably thesubject is a human infected with an alphavirus.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 . RRV-infection results in damages to the cartilage in jointtissues.

20-day-old C57BL/6 mice were infected s.c. With 10⁴ pfu RRV ormock-infected with diluent alone. Infection resulted in extensiveinflammation, pannus formation, articular cartilage thinning, disruptionof the proteoglycans and upregulation of cartilage associated genes. (A,B) For histological analysis the joints of RRV-infected mice weresacrificed at peak disease at 10 days p.i., perfused with 4% PFA. kneejoint tissues removed, paraffin-embedded and 5 µm sections generated.Sections were stained with (A) H&E or (B) Safranin O/fast green.Annotations; (B) bone, (C) cartilage, (P) pannus. Images arerepresentative of at least 5 mice per group (magnification 100X). (C)The width of cartilage from mock- and RRV-infected mice were measured infive areas per mouse and averaged with each point representing onemouse. Cartilage degradation from the medial femoral condyle (MFC) andthe medial tibia plateau (MTP) of RRV mice was assessed as outlined inthe methods, mock control mice show a score of 1. Data represent mean ±SEM of 5 mice per group. (D) Similarly, at both early time points (i)and at peak disease (ii) total RNA from ankle joint tissues was isolatedand analysed for mRNA expression by qRT-PCR. Data was normalised to thehousekeeping gene HPRT1 and expressed as relative expression compared tomock-infected controls (as represented by the dashed line). Each barrepresents the mean +/- the SEM for 5-6 mice per group, *p < 0.05, ***p< 0.001 one-way ANOVA Dunnett’s post test.

FIG. 2 . Pentosan polysulfate reduces the severity of acute RRV-inducedinflammatory disease.

PPS treatment reduced the level of disease signs, prevented severeweight loss and reduced the level of inflammatory infiltrates into thejoint and muscle tissues protecting the muscle tissue from extensiveRRV-induced damage. 20-day-old C57BL/6 mice were infected s.c. with 10⁴pfu RRV or mock-infected with diluent alone then either treated dailyi.p. with PPS at 3 mg/kg in 100 µL PBS or mock-treated with PBS alone.(A) Mice were scored for the development of hind-limb dysfunction anddisplayed a reduction in RRV-disease severity with PPS treatmentMock-infected mice were scored zero for the duration of the experiment*p < 0.05, **p < 0.01 using a Mann-Whitney test. (B) Weight wasmonitored at 24-hour intervals ***p < 0.001 significantly reduced weightloss of RRV-infected PPS-treated compared to RRV-infected mock-treated-using two-way ANOVA with Bonferroni post test. (C) For histologicalanalysis, mice were sacrificed at 7 or 10 days p.i., perfused with 4%PFA, quadriceps and knee joint tissues removed, paraffin-embedded, 5 µmsections generated. Sections were stained with H&E. (D) Quadricepsmuscles were removed from RRV-infected ‘pentosan’ and ‘PBS’ treated miceat day 7 and 10 p.i.. cells were isolated, counted and stained for CD45,Gr1, CD11b, pan NK/NKT, CD3 and CD19 expression. Total leukocyte(CD45^(hi)), inflammatory monocyte (Gr1^(hi)CD1 1 b^(hi)), NK/NKT(CD45^(hi) pan NK^(hi)) and T cell (CD3^(hi)) populations weredetermined among total live (PI-negative) infiltrated cells usingvarious gating strategies, analysed by Student t test. Each data pointrepresents the mean +/-SEM of 5 to 10 mice and is representative of 3-4independent experiments.

FIG. 3 . PPS-treatment does not alter the kinetics of viral replication.

Both infectious virus and viral RNA levels were measured indicatingPPS-treatment did not affect viral clearance. Mice were infected s.c.with RRV or mock-infected with diluent alone then either treated dailyi.p. with PPS or mock-treated with PBS alone. At days 1, 2, 3, 7 and 10p.i. the serum, quadriceps and ankle tissues were harvested, homogenisedand the viral load determined by (A) plaque assay on Vero cells forinfectious virus or (B) by qPCR for viral RNA with nsp3 specific primersusing a standard generated from serial dilutions of RRV T48 infectiousplasmid. Each data point represents a single mouse, line indicates themedian value. *p < 0.05 using two-way ANOVA with Bonferroni post testfor plaque assay and unpaired Mann-Whitney for PCR.

FIG. 4 . PPS-treatment alters the expression of soluble factors inRRV-inflammatory disease.

20-day-old C57BL/6 mice were infected s.c. with 10⁴ pfu RRV ormock-infected with diluent alone then either treated daily i.p. with PPSat 3 mg/kg in 100 µL PBS or mock-treated with PBS alone (mock-infectedmock-treated=Mock control, mock-infected PPS-treated=PPS alone,RRV-infected mock-treated=RRV alone, RRV-infected PPS-treated=RRV+PPS).At days 1, 3 and 10 p.i. serum was collected and analysed for level ofsoluble factors using Bio- Plex Pro Mouse Cytokine 23-plex kits(Biorad). (A) PPS treatment reduced the levels pro- inflammatory factorsat day 10 p.i., (B) increased levels of chemo-attractants factors at day1 p.i. and (C) altered the kinetics of M2 cytokine IL-10. Red lineindicates the level of mock control. Each data point represents the mean+/- standard error of 5 to 6 mice. (A, B) *p < 0.05, ** p < 0.01, ***p <0.001 one-way ANOVA with Tukey’s post test.(C) *p < 0.05, ** p < 0.01,***p < 0.001 using two-way ANOVA with Bonferroni post test. Astricesdirectly on top of bars are compared to mock control levels.

FIG. 5 . Pentosan polysulfate treatment protects the joints fromRRV-induced cartilage damage.

20-day-old C57BL/6 mice were infected s.c. with 10⁴ pfu RRV ormock-infected with diluent alone then either treated daily i.p. with PPSat 3 mg/kg in 100 µL PBS or mock-treated with PBS alone. Forhistological analysis, mice were sacrificed at peak disease at 10 daysp.i., perfused with 4% PFA, whole legs removed, paraffin-embedded and 5µm sections generated. Sections were stained with (A) Masson’s Trichromeor (B) Safranin O/fast green and showed an increase in collagen fibres,improvement in the skeletal muscle tissue morphology and protection ofthe proteoglycan matrix with treatment Annotations: (B) bone. (C)cartilage, (P) pannus, (M) muscle. Images are representative of at least5-8 mice per group. (C) The width of cartilage and epiphyseal plate frommice were measured in five areas of per mouse and averaged with eachpoint representing one mouse. Cartilage degradation from the medialfemoral condyle (MFC) and the medial tibia plateau (MTP) of at leastfive mice per group was assessed as outlined in the methods with mockcontrol mice showing a score of 1. Data represent mean ± SEM of 5 miceper group.

FIG. 6 . Pentosan polysul fate treatment counteracts the dysregulationof the cartilage matrix components caused by RRV-infection.

PPS treatment significantly reduced the early expression of aggrecan(SEQ. ID. NO. 2) and collagen II (SEQ. ID. NO. 4) and the expression ofADAMTS-5 (SEQ. ID. NO. 7) and TIMP-3 (SEQ. ID. NO. 10) at peak disease.20-day-old C57BU6 mice were infected s.c. with 10⁴ pfu RRV ormock-infected with diluent alone then either treated daily i.p. with PPSat 3 mg/kg in 100 µL PBS or mock-treated with PBS alone (mock-infectedmock-treated=Mock control, mock-infected PPS-treated=PPS alone,RRV-infected mock- treated= RRV alone, RRV-infectedPPS-treated=RRV+PPS). At days 1, 3 and 10 p.i. joint tissues wereremoved, RNA extracted and real time PCR performed to evaluate generegulation of key mediators of the proteoglycan matrix of jointcartilage. Results were normalised to the housekeeping gene HPRT1 andare expressed as fold of change compared to the mock control samples.Each data point represents the mean +/- standard error of 5 to 6 miceand is representative of two independent experiments. *p < 0.05. ** p <0.01, ***p <0.001 using two-way ANOVA with Bonferroni post test.

FIG. 7 . Pentosan-polysulfate reduces the severity of acute CHIKV-inflammation without affecting the kinetics of viral infection.

25-day-old C57BL/6 mice were infected s.c. with CHIKV or mock-infectedwith diluent alone then either treated daily i.p. with PPS at 3 mg/kg in100 µL PBS or mock-treated with PBS alone. (A) CHIKV-induced footpadswelling was assessed daily by measuring the height and width of theperimetatarsal area of the hind foot. PPS treatment resulted in asignificant reduction in swelling. (B) H&E stained histological analysisshowed PPS treatment decreased the level of inflammatory infiltrates inCHIKV-infected mouse joints at peak swelling 3 days p.i.. Bothinfectious virus and viral RNA levels were measured indicatingPPS-treatment did not affect viral clearance. At days 1 and 3 and 7 p.i.the serum, quadriceps and ankle tissues were harvested, homogenised andthe viral load determined by (C) plaque assay on Vero cells forinfectious virus or (D) by qPCR for viral RNA in joint tissues withCHIKV E2 specific primers. Each data point represents a single mouse,line indicates the median value. ** p <0.01 using two-way ANOVA withBonferroni post test for foot swelling and plaque assay and Mann Whitneyfor PCR.

FIG. 8 . PPS-treatment alters soluble factors in CHIKV-inflammatorydisease.

25-day-old C57BL/6 mice were infected with CHIKV or diluent alone theneither treated daily i.p. with PPS or mock-treated with PBS alone. (A)Kinetics of IL-10 were altered with PPS-treatment. Grey line indicatesthe level of mock control. Each data point represents the mean +/-standard error of 5 to 6 mice. *p<0.05, ** p< 0.01, ***p < 0.00 1 usingtwo-way ANOVA with Bonferroni post test. Astrices directly on top ofbars are compared to mock control levels. (B) Levels pro-inflammatoryfactors were decreased at peak swelling by day 3 p.i. with PPS treatment*p<0.05, ** p < 0.01, ***p < 0.001 one-way ANOVA with Tukey’s post test.

DESCRIPTION OF EMBODIMENTS

The inflammation arising out of the alphavirus infection is in thejoints and/or muscles. Alternatively, the inflammation may be confinedto the joints or it may be confined to the muscles. In some embodiments,the inflammation will be in both the joints and the muscles.

Although the present invention is directed towards alphavirus infectionsgenerally, in some embodiments, the alphavirus infection will beselected from the group consisting of Ross River virus, chikungunyavirus and Barmah Forest virus.

In particular, the present invention in one embodiment is directedtowards Ross River virus.

In particular, the present invention in another embodiment is directedtowards chikungunya virus.

In particular, the present invention in another embodiment is directedtowards Barmah Forest virus.

Administration of the effective amount of pentosan polysulfate to asubject infected with an alphavirus is parenteral.

In some embodiments when a joint is suffering from inflammation,administration is intra-articular.

In other embodiments when a muscle is suffering from inflammation,administration is intra-muscular.

Generally, a daily dose of pentosan polysulfate will be administered.Such dosing may be into one or more joints, one or more muscles, or intoboth one or more joints and muscles. Alternatively, a single dose may beadministered intravenously to treat inflammation in both joints andmuscles.

It is, however, within the scope of the invention to administer on twomore occasions on a daily basis, depending on the severity of thesymptoms of the subject.

Of course it will be recognised that treatment to reduce cartilagedamage is undertaken by intra-articular or intravenous administration.

Based on the animal studies disclosed herein, the amount to beadministered is within the range of from 0.1 to 5.0 mg/kg body weight ofsubject.

In some embodiments, the amount to be administered is within the rangeof from 1.0 to 5.0 mg/kg body weight of subject.

In some embodiments, the amount to be administered is within the rangeof from 2.0 to 5.0 mg/kg body weight of subject.

Treatment of subjects is by administration to the subject of pentosanpolysulfate. Owing to its solubility and ready availability, preferablythe sodium salt of pentosan polysulfate is used. However, other saltssuch as magnesium and calcium may also be used.

Commercially, Bene-PharmaChem has supplied their PPS in 1 ml glassampoules containing 100 mg PPS/ml. Because of the ready availability ofthis sterile injectable product it is preferred to be used in thepresent invention for treating humans.

The Bene-PharmaChem product comprises: sodium pentosan polysulfate (PPS)100 mg, sodium phosphate 2.2 mg, sodium hydrogen phosphate 6.8 mg,adjusted to pH 6.5 with sodium hydroxide and water for injection, USP,qs. 1 mL.

It is, however, within the scope of this invention to use alternativeformulations. For example, standard textbooks in the field of thisinvention, such as Remington’s Practice of Pharmacy teach suchalternatives.

Likewise, for veterinary applications, a product such as Cartrophen Vet®(Biopharm Australia) may be used.

In order to better understand the nature of this invention, set outbelow is a description of a series of experiments carried out todemonstrate the effectiveness of PPS in the treatment of subjectsinfected with an alphavirus and suffering from joint and/or muscleinflammation and cartilage damage.

METHODS

Virus and Cells. Stocks of the wild-type T48 strain of RRV weregenerated from the full- length T48 cDNA clone (kindly provided by DrRichard Kuhn, Purdue University) (20). Stocks of CHIKV Mauritius strainwere propagated in BHK-21 cells. All titrations were performed by plaqueassay on Vero cells as described previously (21).

Mice. C57BL/6 wild type (WT) mice were obtained from the AnimalResources Centre (Perth. Australia) and bred in- house. All animalexperiments were performed in accordance with the guidelines set out bythe Griffith University Animal Ethics Committee. C57BL/6 mice of twenty-to twenty-five day old, were inoculated subcutaneously (s.c.) with 10⁴pfu virus. Injections of RRV in PBS to a volume of 50 µL was in thethorax as described previously (11), and CHIKV in PBS to a volume of 20µL in the ventral side of the footpad as described previously (10).Mock-infected mice were inoculated with PBS alone. Mice were weighed andscored for disease signs every 24 h. RRV disease scores were assessedbased on animal strength and hind-leg paralysis as outlined previously(22). Swelling of the footpad induced by CHIKV was assessed by measuringthe height and width of the perimetatarsal area of the hind foot usingKincrome digital vernier calipers.

Treatment with pentosan polysulfate (Cartrophen Vet^(®), BiopharmAustralia) or vehicle alone was given intraperitoneally (i.p.) at 3mg/kg in 100 µL PBS (vehicle), daily for the duration of the experiment,commencing from the day of virus infection. In long-term experiments,PPS treatment was delivered orally by adding to drinking water at aconcentration of 100 mg/L which is equivalent to a dose of 25 mg/kg/daybased on the daily water consumption of a C57BL/6 mouse as previouslyreported (23). Using the human to animal conversion outlined byRegan-Shaw et al., (24) 25 mg/kg/day is the equivalent of the human doseof 2 mg/kg which is the recommended dose for Elmiron®.

Histology. Mice were sacrificed and perfused with 4% paraformaldehyde(PFA). Tissues were collected and fixed in 4% PFA, followed by paraffinembedding. Ankles and knee joints were decalcified prior to embedding.Sagittal sections of five micron thickness were prepared and stainedwith haematoxylin and eosin (H & E), Masson’s trichrome or SafraninO/Fast Green. Cartilage thickness and damage was measured at 200Xmagnification from the medial femoral condyle (MFC) and the medialtibial plateau (MTP) by averaging five random points of measurement(separated by at least 20 µM of distance) per region per mouse andgraphed as the mean ± SEM of 5 mice per group. Epiphyseal thickness wasmeasured from central sagittal sections by averaging five random pointsof measurement (separated by at least 20 µM of distance) per region permouse and graphed as the mean ± SEM of 5 mice per group system.Cartilage degradation from the medial femoral condyle (MFC) and themedial tibial plateau (MTP) was assessed according to a modifiedsemiquantitative scoring system of Glasson et al., (25) where 1= normalcartilage; 2= alteration of the proteoglycan matrix assessed by SafraninO stain: 3= alteration of the proteoglycan matrix and loss of laminasplendens; 4= a score of 2-3 thinning either the transitional or radialcartilage; 5= a score of 2-3 plus thinning of both the transitional andradial layers.

Multiplex. The level of serum cytokines was determined using multiplexbead arrays kits according to the manufacturer’s instructions (Bio-PlexPro Mouse Cytokine 23-plex kits) (Biorad, Hercules, CA). Data wasacquired using a Luminex 200™ (Biorad) and analysed using the Bio-plexManager™ 6.1 software (Biorad).

Real-Time PCR. Preparation of RNA was performed from cell pellets usingTRIzol (LifeTechnologies, Victoria, Australia) according to themanufacturer’s instructions. Quantification of total RNA was measured byNanoDrop 1000 spectrophotometer (Thermo Scientific, Victoria,Australia). Extracted total RNA (20 ng/µL) was reverse-transcribed usingan oligo (dT) primer and reverse transcriptase (Sigma Aldrich, Sydney,Australia) according to the manufacturer’s instructions.

Gene expression- SYBR® Green Real-time PCR was performed using 10 ng oftemplate cDNA on a CFX96 TouchTM Real-Time PCR System in 96-well plates,using QuantiTect Primer Assay kits (Qiagen, Hilden, Germany) for HPRT1,or purchased from primers from Sigma-Aldrich with the sequences outlinedin table 1.

TABLE 1 Primer sequences Gene Forward Reverse SEQ. ID.* TGFβ CAA CGC CATCTA TGA GAA AAC C AAG CCC TGTATTCCG TCT CC 1 Aggrecan GCC CAA GAA CAGTAC AAT GGT TGC TAG GTT GGT TGA CCC A 2 Collagen I CAG AAC ATC ACC TACCAC TGC AA TTC AAC ATC GTT GGA ACC CTG 3 Collagen II AGA ACA GCA TCGCCTACC TG CTT GCC CCA CTT ACC AGT GT 4 BMP-⅟mTLD AGC AGG CTG CAG TTC TCAGAC AGC GAA TGT GTT CCG GGC ATA GTG CAT 5 ADAMTS-4 CAC TGA CTT CCT GGACAA TGG TTA T GGA AAA GTC GTC GGT AGA TGG A 6 ADAMTS-5 GAT GAT CAC GAAGAG CAC TAC GA TCA CAT GAA TGA TGC CCA CAT 7 MMP-3 TGG AGC TGA TGC ATAAGC CC TGA AGC CAC CAA CAT CAG GA 8 MMP-9 GGA ACT CAC ACG ACA TCT TCC AGAA ACT CAC ACG CCA GAA GAA TTT 9 TIMP-J GGC ACT CTG GTC TAC ACT ATT AAGCA TTT CAG AGG CTT CCG TGT GA 10 RRV nsp3 primer CCG TGG CGG GTA TTA TCAAT AAC ACT CCC GTC GAC AAC AGA 11 RRV nsp3 Probe ATT AAG AGT GTA GCC ATCC - 12 * A sequence ID listing is attached hereto as Appendix A.

Viral load quantification-Standard curve was generated using serialdilutions of RRV T48 infectious plasmid DNA as described previously(26). Quantification of viral load was performed using SsoAdvancedUniversal Probes Supermix (BioRad) in 12.5 µL reaction volume to detectnsP3 region RNA (table 1) (26).

All reactions were performed using BioRad CFX96 Touch™ Real-Time PCRDetection System on 96-well plates. Cycler conditions were as follows:(i) PCR initial activation step: 95° C. for 15 min, 1 cycle and (ii)3-step cycling: 94° C. for 15 sec, follow by 55° C. for 30 sec and 72°C. for 30 sec, 40 cycles. Dissociation curve was acquired using CFXManager™ software to determine specificity of amplified products.Standard curve was plotted and copy numbers of amplified products wereinterpolated from standard curve using Prism Graphpad software todetermine viral load. The fold change in mRNA expression relative tomock- infected samples for each gene was calculated with the ΔΔCtmethod. Briefly, ΔΔCt = ΔCt (RRV-infected) — ΔCt (Mock-infected) withΔCt = Ct (gene of interest) — Ct (housekeeping gene – HPRT). The foldchange for each gene was calculated as 2^(-ΔΔCt).

Detection of leukocyte infiltrates in quadriceps. Quadriceps muscleswere removed and processed as described previously (10). Briefly,tissues were incubated with 3 mg/Ml collagenase IV and 1 mg/mL DNase Iin 100 µL RPMI 1640 at 37° C. for 1.5 h then resuspended in 5 mL RPMIand passed through a 40 µm cell strainer. Cells were washed, pelletedand treated with 1 × RBC lysis buffer for 5 min, and counted. Todetermine percentages and numbers of specific leukocyte populations,cells were treated with Fc Block (2.4G2; BD) for 5 min at 4° C. andlabelled with fluorochrome-conjugated anti-mouse antibodies, includinganti-CD3-FITC (145-2C11, BD), anti-CD19-APC (MB19-1, eBioscience),anti-CD1 1b-PE (M1/70, BD), anti-Gr1-APC (RB6-8C5, eBioscience) andanti-pan-NK/NKT antigen-PE (U5A2-13, BD) in various combinations in thepresence of biotinylated anti-CD45 (30-F11, eBioscience), followed bytreatment with streptavidin PE-Cy7 at 4° C. for 30 min. Cells wereresuspended in 500 µL PBS containing 2% FCS and 1 µg/mL propidium iodine(PI), and analysed by the CyAn ADP flow cytometer (Beckman Coutler) withKaluza software.

Statistical analysis. Body mass, plaque assay, multiplex (FIGS. 4C and8A), real-time PCR (FIG. 6 ) and joint swelling were analysed usingtwo-way ANOVA with Bonferroni post- test. Real-time PCR (FIG. 1D),multiplex (FIGS. 4B and 8B) and histology (FIG. 5C) were analysed usinga one-way ANOVA with a Dunnett’s or Tukey’s post-test. Flow cytometrydata and histology (FIG. 1C) were analysed using unpaired Studentst-test. All data was tested for normality using the D′Agostino-Pearsonnormality test prior to analysis with these parametric tests. Clinicalscores and and real-time PCR for viral load were analysed using thenon-parametric Mann-Whitney test. Statistics were performed withGraphPad Prism 5.0

RESULTS

Ross River virus infection stimulates the production of proteasesADAMTS-4 (SEQ. ID. NO. 6), MMP-3 (SEQ. ID. NO. 8), and MMP-9 (SEQ. ID.NO. 9) and causes damage to the articular cartilage in joints.

To determine if RRV infection affects the cartilage of joints, weinfected C57BU6 mice with RRV and isolated joint tissue for histologicalanalysis. At peak disease, extensive joint inflammation was observedalong with pannus-like formation and thinning of the articular cartilagein H&E stained sections (FIG. 1A). Further analysis of joint sections,stained with Safranin O, revealed considerable disruption of theproteoglycans in the cartilage matrix, as seen by the difference inSafranin O staining intensity (which is directly proportional to theproteoglycan content) between infected and mock tissues (FIG. 1B) (27).Quantification of cartilage thickness and damage was measured from theMFC and MTP and showed RRV- infection results in an average 20 µmreduction in articular cartilage thickness and cartilage damagecharacterised by alteration of the proteoglycan matrix and loss oflamina splendens (FIG. 1C). Furthermore, RRV-infection resulted in anearly significant increase (24 hours p.i.) of the enzymes: A disintegrinand metalloproteinase with thrombospondin motifs (ADAMTS)-4 (SEQ. ID.NO. 6) (p<0.001), matrix metalloproteinase (MMP)-3 (SEQ. ID. NO. 8)(p<0.05) and MMP-9 (SEQ. ID. NO. 9)(p<0.01) compared to mock infectedcontrols. These are known to cause cartilage damage by degradingaggrecan (SEQ. ID. NO. 2), collagen, proteogylcans and the extracellularmatrix (FIG. 1Di ). At peak disease, stimulators of cartilage growth,matrix-transforming growth factor (TGF) β1 (p<0.01) and bonemorphogenetic protein (BMP)-1 (p<0.001) were also significantlyincreased in response to RRV-infection compared to mock-infectedcontrols (FIG. 1 Dii). The results suggest that RRV infection results incartilage degradation and thinning which is associated with arthriticdisease symptoms.

Pentosan Polysulfate Reduces the Severity of RRV-Induced Disease andInflammation

To assess the potential of PPS as a treatment strategy in alphaviraldisease, mice were infected with RRV or mock-infected with PBS alone andthen treated i.p. with either PPS at 3 mg/kg or with vehicle daily. PPStreatment resulted in a 65 % decrease (p>0.05) in clinical disease scorein RRV-infected mice (FIG. 2A) and a corresponding protection fromdisease-associated weight loss (p>0.001) (FIG. 2B).

To better characterise the reduction in disease, we assessedinflammation and tissue damage in RRV-infected mock and PPS-treatedmice. Tissues from RRV-infected mice were collected at the start and endof peak disease (day 7 and 10 p.i.) for histological analysis and flowcytometry. No inflammation was observed in the quadriceps muscle orankle joint of control mock-infected mock-treated or mock-infectedPPS-treated mice (FIG. 2C). Consistent with previous studies,RRV-infected mock-treated mice showed extensive inflammation andmyositis in quadriceps muscle at day 7 p.i. (FIGS. 2C,D) and around theankle joint (FIG. 2C) (11, 21, 22, 28). In contrast, RRV-infectedPPS-treated mice showed markedly reduced numbers of infiltrating cellsin both the muscle and the joint tissues (FIG. 2C).

In order to characterise the effect of PPS treatment on both lymphoidand myeloid infiltrating cells, we analysed the cell populations in thequadriceps muscles at days 7 and 10 p.i. by flow cytometry. At day 7p.i. PPS treatment uniformly reduced the numbers of all CD45⁺infiltrating leukocytes including reductions (p<0.05) in the monocytes,T cell and NK cell populations (FIG. 2D). By day 10 p.i. PPS treatmentshowed a decrease in the T cell and NK cell populations (p<0.05) withsimilar numbers of CD45⁺ cells and monocytes (FIG. 2D). At day 10 p.i.treatment also altered the NK cell to total cell ratio resulting in areduction in the overall percentage of NK cells in the quadriceps muscle(data not shown).

Reduced Disease in Treated Mice Is Not Due to Decreased Viral Burden

Viral titres in the serum of PPS-treated and mock-treated mice werecomparable at all days tested, indicative of equivalent systemicreplication (FIG. 3A). Similarly, RRV titres recovered from quadricepsmuscles were comparable (FIG. 3A). Interestingly, RRV titres in theankle tissues showed the most variation between PPS-treated andmock-treated mice (FIG. 3A). By day 3 p.i. there was a slight reductionin RRV titres for PPS-treated mice; by day 7 p.i titres were comparable;and then by day 10 p.i. RRV titres in PPS-treated mice were elevatedcompared to mock-treated mice (p<0.05). The results from day 10 p.i.suggest that PPS-treatment may have an effect on viral clearance withinthe joint. To assess viral clearance, qPCR to quantify viral RNA wasperformed on the joint tissues (FIG. 3B). Although the trend appears tosuggest a lower level of specific RRV RNA in the joint tissue ofPPS-treated RRV-infected mice in the joint at ay 10 p.i., this was notstatistically significant compared to mock-treated mice, confirming thatPPS-treatment does not affect viral clearance.

Treatment with pentosan polysulfate increases the level ofanti-inflammatory IL-10 and decreases pro-inflammatory factorsassociated with RRV disease.

A range of pro-inflammatory factors and chemoattractants mediate orcontribute to alphaviral disease (29). To elucidate whetherPPS-treatment affects the production of soluble immune mediators duringRRV infection, sera from PPS-treated and mock-treated mice were analysedusing multiplex and compared to mock-infected, PPS-treated ormock-treated mice. As expected, RRV infection resulted in an increase(p<0.05) of pro-inflammatory factors (both cytokines andchemoattractants) at peak disease (FIG. 4 ). PPS-treatment also alteredthe levels of the M2 anti-inflammatory cytokine IL-10. IL-10 kineticscorresponded to the kinetics of disease with serum levels increasingover time in RRV-infected, mock-treated mice. PPS-treatment resulted inan early surge of IL-10 in RRV-infected mice, being significantlyelevated at both days 1 and 3 p.i. (p<0.001 and 0.01 respectively) (FIG.4A). Additionally, PPS treatment significantly reduced the serum levelsof IL-1α, IL-2, IL-6, CCL-2 and MIP-1α at peak disease (p<0.05) (FIG.4B).

Pentosan polysulfate treatment protects the joints from cartilage damageassociated with RRV infection.

Recently we showed that RRV could infect osteoblasts and infectionresults in systemic bone loss including the tibial epiphysis andvertebrae (30-32). To determine the effect of PPS treatment onRRV-induced muscle and joint damage, the tissues of RRV-infected andmock- infected PPS-treated and mock-treated mice were processed forhistological analysis using Masson’s trichrome and Safranin O/Fast Greenstaining. Masson’s trichrome staining of the tibialis anterior showedPPS treatment protected the morphology of striations within the skeletalmuscle with sections of collagen formation characteristic of musclerepair and fibrosis (33). PPS treatment also prevented RRV-inducedthinning of the epiphyseal plate, protecting against cartilage loss(FIGS. 5A, C). Safranin O/Fast Green staining of the cartilage in theknee joint revealed that PPS treatment protected the proteoglycan matrixof the articular cartilage, preventing the loss of articular cartilageobserved in RRV-infected mock-treated mice as well as maintainingchondrocyte morphology (FIGS. 5B, C).

To further characterise the mechanism of PPS treatment we analysed thegenes involved in enzyme degradation of cartilage (ADAMTS-4 (SEQ. ID.NO. 6), ADAMTS-5 (SEQ. ID. NO. 7), MMP-3 (SEQ. ID. NO. 8), and MMP-9(SEQ. ID. NO. 9)), stimulation of cartilage protection and synthesis(TGF-β1 and BMP-1) and cartilage matrix proteins (aggrecan (SEQ. ID. NO.2), collagen I (SEQ. NO. ID. 3), and collagen II (SEQ. ID. NO. 4)). Asshown in FIG. 1D, RRV-infection caused an increase in ADAMTS-4 (SEQ. ID.NO. 6), which remains elevated at day 3 p.i (p<0.01), but drops by thetime of peak disease (day 10 p.i), where there was a surge in ADMATS-5(SEQ. ID. NO. 7). RRV- infection also resulted in a late rise (at peakdisease) of tissue inhibitor of metalloproteinases (TIMP)-3 (p<0.001):known to inhibit both ADAMTS-4 (SEQ. ID. NO. 6) and ADAMTS-5 (SEQ. ID.NO. 7) (FIGS. 6A, 6C). PPS-treatment significantly reduced the levels ofADAMTS-5 (SEQ. ID. NO. 7) and TIMP-3 (SEQ. ID. NO. 10) at peak disease(p<0.01), but not at the early stages of infection. RRV-infection alsoresulted in an increase of cartilage components aggrecan (SEQ. ID. NO.2), collagen I (SEQ. ID. NO. 3), and collagen II (SEQ. ID. NO. 4), thatis largely reduced with PPS-treatment (FIG. 6B). The genes associatedwith signalling pathways for cartilage development (TGF-β1 and BMP-1)and the metalloproteinases were unaffected by PPS (FIG. 6 ).

Pentosan polysulfate treatment is a safe long-term treatment strategyfor chronic RRV disease.

To assess PPS-treatment for a long-term treatment in patients withchronic symptoms, mice were RRV-infected and treated orally with PPS ormock-treated in drinking water. Long-term PPS treatment resulted in noadverse clinical signs in the mice for the 3-month duration of theexperiment. RRV-infected mock-treated mice showed extended disruption ofthe cartilage components with a three-fold elevation of aggrecan (SEQ.ID. NO. 2) (p<0.01) (Table 2). PPS-treated mice showed less joint damageand significantly decreased the expression of aggrecan (SEQ. ID. NO. 2)back to base-line levels (p<0.001). The levels of ADAMTS-4 (SEQ. ID. NO.6) expression were also reduced (p<0.01).

TABLE 2 Long-term PPS treatment decreases the expression of aggrecan(SEQ. ID. NO. 2) and ADAMTS-4 (SEQ. ID. NO. 6) in RRV infection. MockRRV RRV+PPS Gene Fold Change SEM n Fold Change SEM n Fold Change SEM nADAMTS-4 0.8345 0.2037 6 1.2261 0.2377 5 0.1689** 0.2998 5 ADAMTS-51.0854 0.3209 6 1.5759 0.3730 5 0.8305 0.1796 5 MMP-3 1.1335 0.3492 61.6958 0.4408 5 2.5028 1.2218 5 MMP-9 1.0066 0.0833 6 1.0858 0.2012 50.8604 0.2477 5 Aggrecan 1.0235 0.1579 3 2.9417 0.2865 4 0.6105***0.1050 5 Collagen I 1.0128 0.1178 3 0.8550 0.1417 5 0.4014 0.0748 5Collagen II 1.0357 0.1793 3 0.9584 0.2358 5 0.1719 0.0556 5 TGF-β11.0243 0.1525 3 1.5343 0.2852 5 0.4010 0.0587 5 BMP-1 1.0597 0.2678 31.6204 0.2950 5 1.4697 0.7026 5 TIMP-3 1.3321 0.7729 6 1.4287 0.3184 50.6328 0.1368 5

Pentosan polysulfate is a potential treatment for CHIKV-inducedinflammation, reducing disease by altering the cytokine response.

Given the expanding range of the alphavirus-CHIKV, together with thecurrent lack of therapeutic treatment options, we sought to determinethe broader application of PPS on alleviating CHIKV-induced disease. PPStreatment decreased the level of joint swelling of CHIKV-infected micecorresponding to a reduction in inflammatory cells infiltrating into thejoint (FIGS. 7A, B). Furthermore, as seen in RRV-infection,PPS-treatment did not affect the kinetics of virus infection (FIG. 7C)and did not increase the viral persistence in the joint tissues, withsimilar levels of viral RNA detected three weeks p.i. (FIG. 7D). Thereduced disease also correlated to an early surge in anti-inflammatoryIL-10 (FIG. 8A) and reduced the levels of soluble factors CCL-2, IL-6,IL-9 and G-CSF at peak disease (day 3 p.i.) (FIG. 8B). These collectiveresults, whereby PPS reduces the disease severity of two criticalalphaviral diseases suggest that PPS may be a promising broad-rangetreatment for alphavirus disease manifestations in general.

DISCUSSION

The mechanisms by which alphaviruses trigger arthritis and myositis arethe focus of ongoing studies. Alphavirus-induced disease has manysimilarities to rheumatoid arthritis (RA) including common inflammatorypathways and the key involvement of macrophages (11,12). The innateimmune response is critical in the pathogenesis of alphaviral disease,mediating cell recruitment, viral clearance and inflammation (28, 29,34). In particular, monocytes and macrophages are the major cellularcontributors to disease progression and severity (29). In RA, monocytesplay a significant role in disease development and cartilage destructionthrough the production of pro-inflammatory factors. (35). Despite theseclear similarities, the potential of alphavirus infection to damage thearticular cartilage in the joint tissues has not been investigated. Wenow propose that, analogous to RA, RRV-infection leads to an immunepoly-arthritis that causes cartilage thinning that contributes toclinical signs associated with alphaviral disease.

It has long been recognised that the joint tissue is a critical site ofviral replication, and we recently identified osteoblasts as a source ofinfectious virus, being susceptible to RRV infection (31). We showedthat RRV-infection results in bone loss by disrupting the receptoractivator of nuclear factor κβ ligand (RANKL) and osteoprotegerin (OPG)ratio (31). We now describe thinning of articular cartilage in an RRVdisease mouse model correlating to a significant increase inmetalloproteinases including ADAMTS-4 (SEQ. ID. NO. 6) and ADAMTS-5(SEQ. ID. NO. 7). These result in histopathological findings similar tomild onset RA.

A recent study, using CCR2^(-/-) mice infected with CHIKV, showed thatwhen normal monocyte trafficking is disrupted by this receptor knockoutthe major inflammatory infiltrates became neutrophil dominant (36). Thisreplaces the usual macrophage dominance of the cellular responseobserved in alphavirus infections. Comparing the histopathologicalfindings in the feet of CHIKV-infected wild-type (WT) and CCR2^(-/-)mice, Poo et al (36) observed that the neutrophil shift resulted incartilage damage. We also observe cartilage thinning in immunocompetentC57BL/6 mice (with functional macrophage trafficking) following RRVinfection, demonstrating that macrophages may also play a critical rolein RRV-induced inflammation including cartilage thinning.

The use of glycans as novel therapeutics has developed momentum inrecent years (37). The interaction of glycans with growth factors,extracellular proteases, protease inhibitors, cytokines/chemokines andadhesive proteins regulate various physiopathologies and diseasesincluding cancer, atherosclerosis and thrombosis (38). In addition manypathogens, including viruses, exploit host glycans to cause infection.The therapeutic potential of this class of molecule to alleviateviral-induced arthritis and inflammatory disease has not been studiedand currently remains unknown.

Pentosan polysulfate is a semisynthetic polysaccharide derivative thatchemically and structurally resembles other GAGs, including heparin. Incontrast to many other GAGs, PPS is bioavailable in both its injectableand oral forms and produces limited toxic side effects, even whenadministered in high doses (39). In a clinical setting, PPS has beenused as an anti-thrombotic agent for several decades, due to its abilityto bind preferentially to the glycocalyx of circulating blood cells(40). In more recent times, PPS has been identified as havinganti-inflammatory properties and is currently approved in the UnitedStates for the management of patients with interstitial cystitis, havingan excellent long-term safety profile (15). Furthermore, injectableforms of PPS are currently used to treat osteoarthritis in veterinarymedicine (19).

Given the promising results of PPS treatment of a range of inflammatoryconditions particularly arthritis, and the lack of studies on PPS intreating virus-associated pathologies, we tested the efficacy of PPS totreat alphavirus-induced arthritis. PPS treatment significantly reducedthe acute disease signs and the muscle and joint inflammation of bothRRV- and CHIKV-induced disease. This corresponded to a reduction inserum levels of pro-inflammatory factors at peak disease. In diabetickidney nephropathy, disease pathogenesis is dependent on the cellularinfiltration of macrophages and pro-inflammatory and chemoattractantfactors, similar to those associated with alphavirus-arthritis. Theseinclude CCL-2, RANTES and CXCL1, TNF-α (29, 41). Our data are consistentwith the treatment observed in diabetic nephropathy, in which PPSreduced the macrophage infiltration and suppressed the induction ofpro-inflammatory factors (41).

At the onset of RRV-disease (day 6-7 p.i), there is a surge in thelevels of IL-10 (42). Of interest to the present study is the recentobservation demonstrating that a surge in IL-10 resulted in a phenotypeswitch of monocytes/macrophages (43, 44). This suggests that IL-10 ispart of a repair signal that activates specific cellular and molecularcascades to facilitate tissue recovery (43). PPS-treatment altered thekinetics of RRV-induced soluble pro- and anti-inflammatory factors,promoting a swing to anti-inflammatory cytokines with an early inductionof IL-10 that enhances myogenesis (43). Furthermore IL-10 inhibits thesynthesis of pro-inflammatory soluble factors including, IL-1α, IL-2,IL-6, TNF-α and CCL-2 (45), previously associated with increasedseverity of alphaviral disease. Overall the early PPS- induced increaseof IL-10 may act to reduce inflammation, but also enhance tissue repair,thereby providing a key mechanism by which PPS-treatment reduces theseverity of alphaviral disease.

In addition to the reported action of PPS in reducing pro-inflammatoryfactors, PPS inhibits both the alternative and classical pathways ofcomplement activation (46). For example, in RRV-pathogenesis, complementactivation is essential for the development of severe RRV disease (34).Furthermore, this is specific to RRV-activation of complement via theMBL pathway and is independent of the classical and alternative pathways(28), and therefore it is unlikely that the reduction in RRV diseaseobserved with PPS treatment is due to its effect on complement.

Treatment with PPS also resulted in protection of the i) epiphysis, 2)articular cartilage and 3) proteoglycan matrix. The anti-inflammatoryeffect of PPS is due in part to its ability to inhibit IL-6 (47). In ourresults, serum levels of IL-6 were significantly reduced in PPS treatedRRV- infected mice at peak disease. We have shown that the disruption ofthe RANKL and OPG ratio during RRV-infection occurs in an IL-6 dependentmanner such that inhibition of IL-6 protects from RRV-induced boneloss(31). Additionally, studies on CCL-2 have recently demonstrated thatinhibition of CCL-2 can both inhibit osteoclast differentiation andprotect against CHIKV induced bone loss (30, 48). Therefore, it islikely that PPS protection against RRV-induced bone loss is due to itsability to inhibit both IL-6 and CCL-2. PPS also stimulates hyaluronansynthesis by synovial fibroblasts and proteoglycan synthesis bychondrocytes (49). This may explain the observed protection of both thearticular cartilage and the proteoglycan matrix in PPS-treatedRRV-infected mice. PPS also promotes the proliferation and chondrogenicdifferentiation of adult human bone marrow mesenchymal stem cells (50),further explaining the PPS protection of articular cartilage thinningthat we see in RRV-infection.

Although the molecular mechanism of PPS action remains unclear. PPS canrepress MMP, including ADAMTS expression and inflammation, as well asNF-κB activation. It also enhances proteoglycan synthesis, including theproduction of aggrecan (SEQ. ID. NO. 2) and hyaluronan (49) and has beenshown to be efficacious as both a treatment and a prophylactic (51). Theresults of this study demonstrate that RRV-infection results incartilage thinning, increasing the levels of ADAMTS-4 (SEQ. ID. NO. 6)and ADMATS-5 (SEQ. ID. NO. 7) (aggrecanase 1 and 2 respectively), whichin turn disrupts the proteoglycan matrix in the cartilage, similar tothat reported in osteoarthritis. It also has been demonstrated recentlythat PPS blocks aggrecan (SEQ. ID. NO. 2) breakdown by both bindingdirectly to ADAMTS molecules and inhibiting their action, and byincreasing the affinity between ADAMTS and its inhibitor TIMP-3 (SEQ.ID. NO. 10)(52). We therefore hypothesise that one of the underlyingmechanisms by which PPS treatment prevents cartilage thinning inRRV-induced arthritis, occurs by blocking aggrecan (SEQ. ID. NO. 2)breakdown.

In humans, long-term clinical use of PPS is extremely well tolerated andhighly efficacious for periods greater than 12 months (53). Alphavirusescan produce chronic musculoskeletal ailments over a prolonged period oftime. A long-term therapeutic strategy is therefore required foreffective treatment of alphavirus-induced arthritis. We have shown thatPPS not only alleviates the acute signs of RRV-induced arthritis butalso protects the cartilage over the long-term without compromising hostviral clearance. Given that PPS promotes an anti- inflammatory immunestate without promoting viral persistence, it is an attractivedrug-repurposing candidate for the long-term treatment of RRV-associatedinflammation and disease.

To date, non-steroidal anti-inflammatory drugs are the primarytherapeutic means to alleviate the symptoms of alphavirus-associatedinflammatory disease. These drugs can cause a variety of undesired sideeffects and may compromise immunity in treated patients (54). Studies byour group in the past have examined a number of drug candidates for thetreatment of alphavirus disease. Bindarit (a CCL-2 inhibitor), whileeffective in reducing alphavirus induced arthritis and myositis, iscurrently not a drug that is available for human use (30, 55). Enbrel,while available for human use, was found to suppress the antiviralresponse and enhance viral replication thereby worsen disease (56).Similarly, methotrexate, a licensed drug for the treatment of RA,increased the onset of RRV-induced musculoskeletal disease and theinflux of inflammatory cell infiltrates into the skeletal muscle tissue(57). Here we show PPS treatment significantly reduced both the acuteclinical signs and the inflammation in the muscle (myositis) and thejoint (arthritis) in alphavirus disease. Additionally PPS has positiveand extensive long-term human safety data, and is available as anapproved drug by a number of regulatory authorities globally. Wetherefore conclude that PPS is a promising therapeutic candidate foralphaviral disease; and may also be effective in other infectiousinflammatory conditions.

To this end, the Australian Therapeutic Goods Administration hasprovided its approval for the evaluation of PPS in four Ross River virusinfected subjects. Those subjects were treated with PPS intramuscularlyin accordance with the dosage regimen as described herein.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the above-describedembodiments, without departing from the broad general scope of thepresent disclosure. The present embodiments are, therefore, to beconsidered in all respects as illustrative and not restrictive.

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1. A method of treating a subject suffering from arthralgia resultingfrom an arthralgia causing alphavirus infection comprising administeringby the parenteral route an amount of pentosan polysulfate, or apharmaceutically acceptable salt thereof, effective to reduce the painof the arthralgia in a joint or muscle of the subject without affectingthe viral clearance in that joint.
 2. The method of claim 1 wherein theinflammation is in the joints and/or muscles.
 3. The method of claim 2wherein the inflammation is in the joints.
 4. The method of claim 2wherein the inflammation is in the muscles.
 5. The method of claim 2wherein the inflammation is in the joints and the muscles.
 6. The methodof claim 1 wherein the arthralgia causing alphavirus is selected fromthe group consisting of Ross River virus, chikungunya virus and BarmahForest virus.
 7. The method of claim 6 wherein the virus is Ross Rivervirus.
 8. The method of claim 6 wherein the virus is chikungunya virus.9. The method of claim 6 wherein the virus is Barmah Forest virus. 10.The method of claim 1 wherein administration is intra-articular.
 11. Themethod of claim 1 wherein administration is intra-muscular.
 12. Themethod of claim 1 wherein the pentosan polysulfate is the sodium salt.13. The method of claim 1 wherein administration is daily.
 14. Themethod of claim 1 wherein administration is twice daily.
 15. The methodof any one of claim 1 wherein the amount administered is within therange of from 0.5 to 5.0 mg/kg body weight of subject.
 16. The method ofclaim 15 wherein the amount is within the range of from 1.0 to 5.0 mg/kgbody weight of subject.
 17. The method of claim 16 wherein the amount iswithin the range of from 2.0 to 5.0 mg/kg body weight of subject. 18.The method of claim 17 wherein the subject is a human.
 19. A method oftreating a human subject suffering from arthralgia resulting from anarthralgia causing alphavirus infection comprising administering by theparenteral route an amount of sodium pentosan polysulfate within therange of from 2.0 to 5.0 mg/kg body weight of the subject, so as toeffectively reduce the pain of the arthralgia in a joint or muscle ofthe subject without affecting the viral clearance in that joint.
 20. Themethod of claim 19 wherein the virus is Ross River virus.
 21. The methodof claim 19 wherein the virus is chikungunya virus.
 22. The method ofclaim 19 wherein the virus is Barmah Forest virus.